Given the increasing evidence that RNA binding proteins (RBPs) serve as genetic causes or predisposing risk factors underlying a wide spectrum of neurological diseases, a pressing need exists to understand their specific cellular functions in developing and mature brains. Similar to transcription factors, RBPs could be master regulators of certain cellular processes owing to their coordinated target sets. Unlike widely-studied transcription factors, the physiological functions of RBPs are historically underexplored. Most of the dozen RBPs found to exhibit tissue-specific expression are involved in regulating neuronal alternative splicing. We and others have revealed the large-scale genetic programming of alternative splicing during embryonic brain development, resulting in neuron-specific alternative isoforms in mature brains. We show many of these splicing changes are mediated by a nuclear RBP, polypyrimidine tract-binding protein 2 (PTBP2). PTBP2 exhibits dynamic temporal expression during neuronal differentiation and may orchestrate the developmental programming of alternative splicing in order to accomplish the prolonged and continuous neuronal morphological transformations. Our previous study shows that downregulation of PTBP2 prior to synaptogenesis is necessary for spine formation. Our newest data show that PTBP2 has additional functions in early differentiating neurons prior to synapse formation. Guided by strong preliminary data, we hypothesize that PTBP2 governs neuron-specific splicing to control the initiation of neuronal polarity. This proposal will address multiple critical barriers to the study of neuronal polarity and provide a new framework for functional analysis of alternative splicing. Our long history of researching alternative splicing and PTBP2 in the brain places us in a unique position to advance these fields. Our team, with complementary expertise in genetics, neurobiology, and molecular cellular biochemical and computational biology, has demonstrated successful collaborations. We have generated new tools and resource to thoroughly determine the cellular and molecular defects caused by Ptbp2 knockout in cortical neurons. Completion of this project will allow us to further our long-term goal of revealing new genetic, molecular and cellular controls of neuronal polarity and morphogenesis that enables neural circuit formation.